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Modified annexin proteins and methods for preventing thrombosis

a technology of annexin proteins and thrombosis, which is applied in the direction of drug compositions, peptides, cardiovascular disorders, etc., can solve the problems of increased intracerebral hemorrhage risk, increased risk of cerebral hemorrhage, and no benefit of antibody, so as to prevent arterial or venous thrombosis without increasing hemorrhage, prolong the half-life in the vascular compartment, and prevent thrombosis

Inactive Publication Date: 2005-11-08
SURROMED
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

"The present invention provides compounds and methods for preventing blood clots without increasing the risk of bleeding. This is achieved by modifying a protein called annexin, which prevents the formation of a protein complex that triggers blood clotting. The modification can be done by adding a polymer or another protein to the annexin. The modified annexin binds to phosphatidylserine on the surface of activated platelets or injured cells, preventing the formation of a prothrombinase complex. The invention also provides pharmaceutical compositions containing the modified annexin proteins for preventing thrombosis. The modified annexin can be administered to patients at risk of thrombosis, such as those with diabetes or pregnancy. The invention also includes a method for screening for modified annexin proteins and a method for in vivo screening in animals."

Problems solved by technology

However, bolus injection (as opposed to a more gradual intravenous infusion) significantly increases the risk of cerebral hemorrhage (Mehta et al., 2000).
However, the same antibody has shown no benefit in unstable angina without angioplasty, and a better method for preventing coronary occlusion in these patients is needed.
However, intravenous rtPA administration is associated with increased risk of intracerebral hemorrhage.
Venous thrombosis is a frequent complication of surgical procedures such as hip and knee arthroplasties.
Despite such promising results for preventing thrombosis, a major problem associated with the therapeutic use of annexins is their short half-life in the circulation, estimated in experimental animals to be 5 to 15 minutes (Römisch et al., 1991; Stratton et al., 1995; Thiagarajan and Benedict, 1997); annexin V also has a short half-life in the circulation of humans (Strauss et al., J.

Method used

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  • Modified annexin proteins and methods for preventing thrombosis
  • Modified annexin proteins and methods for preventing thrombosis

Examples

Experimental program
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example 1

Modified Annexin Preparation

[0089]Annexins can be purified from human tissues or produced by recombinant technology. For instance, annexin V can be purified from human placentas as described by Funakoshi et al. (1987). Examples of recombinant products are the expression of annexin II and annexin V in Escherichia coli (Kang, H.-M., Trends Cardiovasc. Med. 9:92-102 (1999); Thiagarajan and Benedict, 1997, 2000). A rapid and efficient purification method for recombinant annexin V, based on Ca2+-enhanced binding to phosphatidylserine-containing liposomes and subsequent elution by EDTA, has been described by Berger, FEBS Lett. 329:25-28 (1993). This procedure can be improved by the use of phosphatidylserine coupled to a solid phase support.

[0090]Annexins can be coupled to polyethylene glycol (PEG) by any of several well-established procedures (reviewed by Hermanson, 1996) in a process referred to as pegylation. The present invention includes chemically-derivatized annexin molecules having...

example 2

In Vitro and In Vivo Assays

[0098]In vitro assays determine the ability of modified annexin proteins to bind to activated platelets. Annexin V binds to platelets, and this binding is markedly increased in vitro by activation of the platelets with thrombin (Thiagarajan and Tait, 1990; Sun et al., 1993). Preferably, the modified annexin proteins of the present invention are prepared in such a way that perform the function of annexin in that they bind to platelets and prevent protein S from binding to platelets (Sun et al., 1993). The modified annexin proteins also perform the function of exhibiting the same anticoagulant activity in vitro that unmodified annexin proteins exhibit. A method for measuring the clotting time is the activated partial thromboplastin time (Fritsma, in Hemostasis and thrombosis in the clinical laboratory (Corriveau, D. M. and Fritsma, G. A., eds) J. P. Lipincott Co., Philadelphia (1989), pp. 92-124, incorporated herein by reference).

[0099]In vivo assays determi...

example 3

[0100]The anticoagulant ability of human recombinant annexin V and pegylated human recombinant annexin V were compared in vitro.

[0101]Annexin V production. The polymerase chain reaction was used to amplify the cDNA from the initiator methionine to the stop codon with specific oligonucleotide primers from a human placental cDNA library. The forward primer was 5′-ACCTGAGTAGTCGCCATGGCACAGGTTCTC-3′ (SEQ ID NO:7) and the reverse primer was 5′-CCCGAATTCACGTTAGTCATCTTCTCCACAGAGCAG-3′ (SEQ ID NO:8). The amplified 1.1-kb fragment was digested with Nco I and Eco RI and ligated into the prokaryotic expression vector pTRC 99A. The ligation product was used to transform competent Escherichia coli strain JM 105 and sequenced.

[0102]Recombinant annexin V was isolated from the bacterial lysates as described by Berger et al., 1993, with some modification. An overnight culture of E. coli JM 105 transformed with pTRC 99A-annexin V was expanded 50-fold in fresh Luria-Bertrani medium containing 100 mg / L ...

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Abstract

A modified annexin protein, preferably annexin V, is used to prevent thrombosis without increasing hemorrhage. Annexin binds to phosphatidylserine on the outer surface of cell membranes, thereby preventing binding of the prothrombinase complex necessary for thrombus formation. It does not, however, affect platelet aggregation necessary for hemostasis. The modified annexin molecule can be a homodimer of annexin, an annexin molecule coupled to one or more polyethylene glycol chains, or an annexin molecule coupled to another protein. By increasing the molecular weight of annexin, the modified annexin is made to remain in circulation for sufficient time to provide a sustained therapeutic effect.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS[0001]This application claims priority from U.S. Provisional Application No. 60 / 270,402, “Optimizing the Annexin Molecule for Preventing Thrombosis,” filed Feb. 21, 2001, and U.S. Provisional Application No. 60 / 332,582, “Modified Annexin Molecule for Preventing Thrombosis and Reperfusion Injury,” filed Nov. 21, 2001, both of which are incorporated herein by reference.FIELD OF THE INVENTION[0002]The present invention relates generally to methods and compositions for treating thrombosis. More particularly, it relates to modified annexin proteins and methods for their use.BACKGROUND OF THE INVENTION[0003]Thrombosis—the formation, development, or presence of a blood clot (thrombus) in a blood vessel—is the most common severe medical disorder. The most frequent example of arterial thrombosis is coronary thrombosis, which leads to occlusion of the coronary arteries and often to myocardial infarction (heart attack). More than 1.3 million patients are ...

Claims

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Application Information

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Patent Type & Authority Patents(United States)
IPC IPC(8): A61K38/17A61K47/48A61K38/00G01N33/50A61P7/02A61P9/10C07K14/47C07K16/18C07K19/00C12N1/15C12N1/19C12N1/21C12N5/10C12N15/09C12P21/02C12Q1/00G01N33/15
CPCA61K38/1709A61K47/48215A61K47/48238A61K47/60A61K47/62A61P7/02A61P9/10
Inventor ALLISON, ANTHONY
Owner SURROMED
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